Competition among host‐specific lineages of Poecilochirus carabi mites influences the extent of co‐adaptation with their Nicrophorus vespilloides burying beetle hosts

Abstract Reciprocal selection between symbiotic organisms and their hosts can generate variations in local adaptation between them. Symbionts often form species complexes with lineages partially adapted to various hosts. However, it is unclear how interactions among these lineages influences geographic variation in the extent of host‐symbiont local adaptation. We addressed this shortcoming with experiments on burying beetles Nicrophorus vespilloides and their specialist phoretic mite Poecilochirus carabi in two adjacent woodlands. Burying beetles transport these mites to vertebrate carrion upon which they both reproduce. P. carabi appears to be a species complex, with distinct lineages that specialise on breeding alongside different Nicrophorus species. We found that in one wood (Gamlingay Woods), N. vespilloides carries a mixture of mite lineages, with each lineage corresponding to one of the four Nicrophorus species that inhabits this wood. However, two burying beetle species coexist in neighbouring Waresley Woods and here N. vespilloides predominantly carries the mite lineage that favours N. vespilloides. Mite lineage mixing alters the degree of local adaptation for both N. vespilloides and the P. carabi mites, affecting reproductive success variably across different woodlands. In Gamlingay, mite lineage mixing reduced N. vespilloides reproductive success, while experimentally purifying mites lineage enhanced it. The near pure lineage of vespilloides mites negligibly affected Waresley N. vespilloides. Mite reproductive success varied with host specificity: Gamlingay mites had greatest reproductive success on Gamlingay beetles, and performed less well with Waresley beetles. By contrast, Waresley mites had consistent reproductive success, regardless of beetle's woodland of origin. We conclude that there is some evidence that N. vespilloides and its specific mite lineage have coadapted. However, neither N. vespilloides nor its mite lineage adapted to breed alongside other mite lineages. This, we suggest, causes variation between Waresley and Gaminglay Woods in the extent of local adaptation between N. vespilloides beetles and their P. carabi mites.


| INTRODUC TI ON
Ever since Darwin (1859), evidence has been gathering that natural selection causes populations to adapt in different ways to their different local environments.More recent work suggests that adaptation can happen on very local scales, even when there is still gene flow between populations (e.g.Sun et al., 2020;Thompson, 2013).
Nevertheless, the extent of local adaptation varies across landscapes, between populations of the same species (e.g.Thompson, 1999Thompson, , 2005)).This is especially true for adaptations that arise from coevolutionary interactions between symbiotic species (by which we mean 'intimately coexisting' species), which are engaged in reciprocal selection.In specialist antagonistic interactions, or obligate mutualisms, each party exerts such strong selection on the other that there can be little differentiation between populations (e.g.Forde et al., 2004;Johnson et al., 2010;Lively & Dybdahl, 2000).
A key challenge is to understand the ecological factors that generate such geographical mosaics of co-evolution and co-adaptation (Thompson, 2013).
Environmental differences among populations are known to cause variation in the outcome of any species interaction.These might arise from variation in the availability of an essential resource (e.g.Johnson et al., 2010), or the presence of a common enemy species (e.g.Hopkins et al., 2017;Sun & Kilner, 2020) or differences in the abiotic environment (e.g.Kersch & Fonseca, 2005).Theoretical analyses suggest that this variation, in turn, can cause dramatic differences in the trajectory of local co-evolution (e.g.Nuismer et al., 2000).Therefore, determining ecological correlates of variation in the extent of reciprocal selection between the same two partner species can potentially explain why there is geographical variation in the pattern of local adaptation among populations (e.g.Garrido et al., 2012;Gorter et al., 2016;Johnson et al., 2010).
However, in reality, interactions between species rarely involve just two partner species.For example, one insect species commonly pollinates more than one plant species, while each plant species can be pollinated by more than one insect species (e.g.Thompson, 2013).
Similarly, one cactus species can be in a protective mutualism with multiple species of ants (Ness et al., 2006), while multiple bumblebee species Bombus spp.commonly interact with multiple mite species (Haas et al., 2019).Likewise, host species richness and abundance are positively correlated with parasite species richness (e.g.Hechinger & Lafferty, 2005).Multispecies associations are likely to generate variation in the strength of reciprocal selection.For example, multiple infection of different parasite species or strains can differentially influence the fitness among different host species interacting in the same community, depending on the susceptibility and tolerance of each host species (Friesen et al., 2017).Therefore understanding interactions among different strains or species of symbionts is likely to help account for geographical variation in the pattern of local coadaptation between symbionts and their hosts.
Here we apply this rationale to understanding the extent of local co-adaptation between burying beetles Nicrophorus vespilloides (Coleoptera: Silphidae) and the species complex of Poecilochirus carabi mites with whom they interact (Mesostigmata: Parasitidae).
Both the burying beetle and the mite require small vertebrate carrion to breed, but only the beetle is capable of flight.The mites travel with the beetle between breeding opportunities while sexually immature deutonymphs, attached as benign passengers to the adult beetle's thorax.When they locate a dead body, the adult beetles convert the carrion into an edible nest for their young, and then stay with their brood to defend and nourish them.Meanwhile, the mites disembark, moult into the adult stage, mate and produce offspring alongside the beetle larvae (Schwarz & Müller, 1992).Breeding alongside mites can be costly for beetles (De Gasperin et al., 2015;De Gasperin & Kilner, 2015;Nehring et al., 2019), which select adaptations in the beetles to mitigate these costs.When their duties of care are complete, adult beetles fly off in search of new reproductive opportunities, carrying with them the next generation of mites.The timing of the mite's lifecycle is therefore closely aligned with the duration of beetle parental care (Schwarz & Müller, 1992).
Poecilochirus carabi exists as a species complex because it comprises distinct lineages of mites that are each specialised to breed on different species of burying beetle Nicrophorus spp.(Nehring et al., 2017).Each burying beetle species differs slightly in its duration of care.This has favoured divergent adaptation in the mite populations associated with each species of burying beetle, which in turn has generated distinct mite lineages (Canitz et al., 2021;Müller & Schwarz, 1990;Wilson, 1982).Nevertheless, mite lineages can still interbreed and cannot be told apart morphologically.The key way to distinguish them is behaviourally, through their relative preference for different burying beetle species (Brown & Wilson, 1992;Schwarz, 1996;Wilson, 1982).Previous work suggests that when sympatric Nicrophorus species do not differ much in their duration of care, then each species of burying beetles carries a mixture of the different mite lineages associated with each of the sympatric beetles and the mites hybridise across lineages (Brown & Wilson, 1992).

T A X O N O M Y C L A S S I F I C A T I O N
Behavioural ecology, Biodiversity ecology, Community ecology, Entomology, Evolutionary ecology especially when larger beetle species have relatively longer larval development time than smaller beetle species, each species is more likely to carry its own specialist mite lineage and each mite lineage is more likely to be reproductively isolated (Brown & Wilson, 1992).
We focused on N. vespilloides and P. carabi inhabiting two neighbouring yet geographically isolated populations (Gamlingay and Waresley Woods) in Cambridgeshire, United Kingdom.Previous studies have shown that there are differences in the Nicrophorus guild inhabiting each woodland (Sun et al., 2020).Both woods contain the smallest UK burying beetle N. vespilloides and the larger burying beetle Nicrophorus humator.However, only Gamlingay Woods is additionally routinely inhabited by the intermediate-sized Nicrophorus interruptus and Nicrophorus investigator.We tested the following predictions that 1. N. vespilloides from Gamlingay Woods should be more likely to carry a mixture of mite lineages, whereas N. vespilloides from Waresley Woods should be more likely to carry the N. vespilloides lineage of mites, reflecting the different species that routinely coexist in each wood.To test this prediction, we conducted two consecutive choice experiments for mites to choose among all four Nicrophorus beetles in the first generation of P. carabi, and then bred these mites based on their choice.Their offspring were tested again to evaluate the consistency in choice and the extent to which each mite lineage was mixed in each woodland.

N. vespilloides should have lower reproductive success when
breeding alongside a mixture of P. carabi lineages than when breeding with its specialist P. carabi lineage, with whom the beetle is more likely to be co-adapted.We experimentally manipulated the mite composition, either mixing all four lineages of P. carabi or allowing the pure N. vespilloides lineage of P. carabi to breed alongside N. vespilloides.At dispersal, we determined the reproductive success of both beetles and mites.

N. vespilloides and P. carabi should be divergently co-adapted in
Gamlingay and Waresley woods, if they differ in whether they carry pure versus mixed lineages of mites.Specifically, we predicted that N. vespilloides and its specialist lineage of mites should be locally adapted to each other.We tested this prediction using reciprocal transplant experiments by exposing mites from either Gamlingay Woods or Waresley Woods to N. vespilloides beetles from Gamlingay and Waresley Woods.Just as before, we determined the reproductive success of both beetles and mites at dispersal.

| Study species
Gamlingay and Waresley Woods are fragments of the Wild Wood that covered England until deforestation (c.1000-4000 years ago), and are roughly 2.5 km apart (Sun et al., 2020).N. vespilloides are the most abundant burying beetles in each wood (81.6% in Gamlingay Woods and 93.7% in Waresley Woods (Sun et al., 2020)) and the P. carabi mite species complex is the most commonly found mite species associating with Nicrophorus beetles (Schwarz et al., 1998).

| Field observations
Surveys of burying beetle communities were conducted in Gamlingay (Latitude: 52.15555°; Longitude: −0.19286°) and Waresley (Latitude: 52.17487°; Longitude: −0.17354°) Woods in Cambridgeshire, United Kingdom.From June to October in 2016-2017, five traps at each site were baited with mouse carcasses and hung in the same location, separated by at least 150 m.We checked the traps every 2-3 weeks and collected all Nicrophorus spp.under permit.Traps were then refilled with fresh mice at each collection.The number and sex of each beetle species were recorded at each location for each trapping event.Each beetle's pronotum width was measured to the nearest 0.01 mm as a standardised measurement of body size (Jarrett et al., 2017).We separated mites from their beetle hosts under anaesthetic by CO 2 .The data presented here are related to those from an earlier published study within this system that focussed only on the correlation between body size and mite load in N. vespilloides in 2017 (Sun et al., 2019).In the current study, we have used a more extensive dataset to thoroughly investigate the mite load on each Nicrophorus spp.during 2016 and 2017.

| Origin and maintenance of burying beetles and mites
Both species were kept under laboratory conditions at 21 ± 2°C and on a 16:8 light to dark cycle.
Beetles were kept individually in plastic boxes (12 cm × 8 cm × 2 cm) filled with moist soil.Field-collected beetles were kept for at least 2 weeks before they were subjected to experimentation to even out any differences in sexual maturity and nutritional status.To breed beetles, N. vespilloides collected from the field sites were paired on a mouse in a breeding box lined with damp soil.All breeding boxes were then placed into cupboards to mimic underground environments.After 8 days, we collected the dispersing larvae and transferred them to eclosion boxes (10 × 10 × 2 cm, 25 compartments) filled with moist soil.At eclosion, each emerging beetle was moved to a plastic container (12 × 8 × 2 cm) with moist soil.We fed beetles twice a week with minced beef for 2-3 weeks until they were sexually mature.
Mites were maintained in distinct populations, according to their woodland of origin, and kept apart from burying beetles except when breeding.To breed mites, each month we transferred 15 mite deutonymphs chosen at random, and a pair of beetles from the same population, to a new breeding box (17 × 12 × 6 cm with 2 cm of soil) furnished with a fresh mouse carcass (n = 10 for each population).After breeding, beetle parents and third-instar larvae were removed from the box.The mites remained and were given another adult beetle, and thereafter supplied with minced beef twice a week.

| Prediction 1: Gamlingay N. vespilloides carry a mixture of P. carabi lineages
To investigate population differences in the number of mite lineages present in each wood, we used consecutive choice experiments.Longitude: 0.04303°) and Thetford Forest (Latitude: 52.41286°; Longitude: 0.75167°)).This allowed us to remove any beetle population-level effect on mite preferences, and to focus entirely on the effect of the beetle's species on influencing mite preferences.
We used mites that had been bred for one generation in the lab so that the tested mites had no prior experience which might influence their choice of beetle.Four burying beetles (of the same sex, one from each species; n = 43 trials where beetles were all males and 38 trials where beetles were all females) were introduced into a plastic container (17 × 12 × 6 cm), around which they could move freely.
At the same time 50 mites were also introduced-either from the Gamlingay population or from the Waresley population.It is unlikely that the mites hindered each other in attaching to host beetles because wild-caught beetles can carry up to 200-300 mites individually, even on N. vespilloides, the smallest of the UK species (Schwarz et al., 1998;Sun et al., 2019).The container held moist soil to a depth of 2 cm and minced beef in ad libitum quantities.All beetles and mites were starved for a day prior to the experiment to increase the likelihood that they gathered together on the mince to mimic the natural context in which mites transfer between beetles hosts at feeding/ breeding opportunities.The number of mites carried by each beetle species was recorded 24 h later, and used to assess the mixing of different mite lineages in each population.Mites that chose N. vespilloides, N. humator, N. interruptus and N. investigator were defined as P-ves, P-hum, P-int and P-inv, respectively.We then bred these mites separately on a fresh mouse carcass, with one mouse carcass for each lineage of mite identified by the choice test.The offspring of these breedings were then tested again for their burying beetle preferences, as a further test of extent to which the mite lineages were mixed in each woodland.For this second choice test, we introduced 10 mites and one beetle from each of the four species of the same sex (n = 92 trials where beetles were all males and 50 trials where beetles were all females) in a plastic container, and counted the number of mites on each beetle after 24 h.We tested 10 mites in the second choice experiment because we harvested relatively few P-hum and P-inv in the first choice experiment, and this meant there were fewer offspring available for testing.

| Prediction 2: A mixture of P. carabi lineages negatively affects N. vespilloides
These experiments were focused on N. vespilloides and P. carabi mites drawn from Gamlingay Woods.We experimentally manipulated the composition of the mite community (= a group of 10 deutonymphs) associated with each burying beetle, generating two treatments: (a) pure N. vespilloides lineage of P. carabi and (b) a mixture of all four lineages of P. carabi.In the mixture, we introduced 3 P-ves, 1 P-hum, 4 P-int and 2 P-inv, based on the relative proportion of the four P. carabi lineages from Prediction 1.The mites used were descendants of the second generation of P. carabi from the experiment above, and lineages were determined from the preferences they exhibited in the second generation of choice experiment.Ten mites were introduced at beetle pairing, directly onto the carcass.Pairs of beetles were sequentially assigned to one of the three mite treatments, introduced into a breeding box (17 × 12 × 6 cm with 2 cm of soil) and given a 15-20 g (17.71 ± 0.16) mouse carcass to breed upon.At larval dispersal, that is, 8 days after pairing, we counted all larvae and weighed the whole brood (to the nearest 0.1 mg).We calculated the average larval mass for each brood (total brood mass divided by the number of larvae).To determine the reproductive success of mites, we used CO 2 to detach dispersing mite deutonymphs from adult beetles, at the end of the breeding event.

| Prediction 3: Populations have adapted divergently
To assess the extent of local adaptation, we adopted a fully factorial design of experimental reciprocal mite infestation (e.g.Blanquart et al., 2013;Garrido et al., 2012;Nuismer & Gandon, 2008).These experiments were carried out in three blocks.Each beetle population (Gamlingay/Waresley) was infested with either 10 Gamlingay mite deutonymphs or 10 Waresley mite deutonymphs.Mites were introduced directly onto 15-20 g carcasses (16.97 ± 0.12 g), when beetles were paired.Pairs of beetles were unrelated, to prevent inbreeding.We took the same measurements of beetle and mite reproductive success as in the previous experiments, when larvae dispersed away from the carcass 8 days after pairing.

| Statistical analyses
We analysed the data using generalised linear mixed models (GLMM) with the glmer function in the lme4 package in R version 3.4.3(R Development Core Team, 2014).To obtain minimal adequate models, we applied a stepwise approach to exclude non-significant variables and interactions (Crawley, 2007).We included block as a random effect in all models, where appropriate.Tukey HSD tests were used for post hoc pairwise comparisons, as necessary, using the lsmeans package (Lenth, 2016).All data are provided in Appendix S1.

| Prediction 1: Gamlingay N. vespilloides carry a mixture of P. carabi lineages
We analysed variation in the number of mites carried by each beetle species using a Poisson model, with an offset of the log total number of mites allowed to make a choice in each trial.We included mite population (Gamlingay/Waresley), beetle species (N.vespilloides, N. humator, N. interruptus and N. investigator) and their interaction as explanatory variables.Sex and body size of beetles were included as covariates.We also included as random factors the sampling year (2017 or 2018), and trial ID.We analysed the data in a similar manner for the second-choice experiment, testing for consistency of beetle species preference among mites in the next generation.Of the 16 possible outcomes this two-generation experiment yielded (N = 4 species for mites to choose in Gen 1 × N = 4 species for mites to choose in Gen 2), we analysed only those the mites that made the same choice of beetle species as their parents (i.e.N = 4 outcomes).

| Prediction 2: A mixture of P. carabi lineages negatively affects N. vespilloides
We used generalised linear models (GLM) to analyse three measures of beetle reproductive success when exposed to different experimental combinations of mite lineages: brood size (using a Poisson distribution), brood mass and average larval mass (both using a Gaussian distribution).We included mite treatment as an explanatory variable, and additionally included carcass mass as a covariate when analysing brood size and brood mass.For analysis of average larval mass, we included larval density (brood size divided by carcass mass) as a covariate.We also analysed variation in mite reproductive success using a negative binomial GLM with mite treatments and carcass mass as explanatory variables.

Local adaptation of beetles to mites
We analysed three measures of beetle reproductive success when exposed to different mite populations using GLMMs: brood size, brood mass and average larval mass.Beetle wood of origin (Gamlingay/Waresley), mite wood of origin (Gamlingay/Waresley) and their interaction were included as explanatory variables.We included carcass mass as a covariate for the analysis of brood size and brood mass, whereas we included larval density (brood size divided by carcass mass) as a covariate for the analysis of average larval mass.In all models, block was included as a random factor.

Local adaptation of mites to beetles
We analysed the number of dispersing mites deutonymphs present at the end of each reproductive bout using a negative binomial GLMM.Beetle wood of origin (Gamlingay/Waresley), mite wood of origin (Gamlingay/Waresley) and their interaction were included as explanatory variables.Carcass mass was included as a covariate, whereas block was included as a random factor.

| Field observation
In total, 1465 Nicrophorus individuals were caught over the two sampling years (780 and 685 for Gamlingay and Waresley Woods, respectively), carrying a total of 17,249 P. carabi mite deutonymphs on four beetle species (Figure 1).We found that the mite load on each Nicrophorus species varied differently between populations (beetle species × population interaction, χ 2 = 46.19,df = 3, p < .001; Figure 1).Specifically, N. vespilloides from Gamlingay had an average lower number of mites compared to N. interruptus carried more mites than N. humator (post hoc comparison: z = 6.80, p < .001),but we could detect no difference in the mite load carried by N. vespilloides and N. interruptus (post hoc comparison: z = 2.22, p = .118),nor between N. vespilloides and N. investigator (post hoc comparison: z = 1.14, p = .663).Moreover, comparing mite abundance on the beetle species, between woodlands, we found that Gamlingay N. vespilloides had lower number of mites than Waresley N. vespilloides (post hoc comparison: z = −4.18,p < .001).In contrast, N. humator from Gamlingay had higher mite abundance than N. humator from Waresley (post hoc comparison: z = 4.45, p < .001),and there was a tendency for Gamlingay N. interruptus to carry more mites than those from Waresley (post hoc comparison: z = 1.72, p = .086).We could detect no significant difference between Gamlingay and Waresley in mite abundance on N. investigator (post hoc comparison: z = 0.90, p = .370).
We used post hoc comparisons to compare the strength of the mite preference for each Nicrophorus species between populations.We found that Gamlingay mites showed stronger preference for N. humator (z = 5.99, p < .001)and N. interruptus (z = 5.45, p < .001)than mites from Waresley.They were also slightly inclined to favour N. investigator, but this pattern was not statistically significant (z = 1.93, p = .054).By contrast, Waresley mites showed a higher preference for N. vespilloides than Gamlingay mites (z = −9.04,p < .001).
We bred mites that showed the same preference for burying beetle species, and tested whether the preferences of the offspring matched those of their parents, to test for indirect evidence that mites were segregating into genetic lineages, as inferred from their host preference.The extent to which the beetle preferences aligned between the generations varied by woodland (mite population × beetle species interaction, χ 2 = 50.42,df = 3, p < .001).We found that Waresley P-ves mites consistently had stronger preference for N. vespilloides than Gamlingay mites (t = −2.17,p = .030;

vespilloides
We tested whether differential mixing of mite lineages in Gamlingay Woods caused variations in beetle reproductive success.We created experimental mite communities, manipulated to different degrees to contain mites with different beetle preferences.

Brood size
Beetles exposed to Gamlingay and Waresley mites produced a similar brood size (Figure 4a; Table 2).In addition, beetles from Waresley Woods consistently produced larger broods compared to those from Gamlingay Woods (Figure 4a; Table 2).

TA B L E 1
Results of Tukey's post hoc comparisons for beetle species × population interaction in the first choice experiment.

TA B L E 2
Results from the final models analysing the fitness components of local adaptation between beetles and mites.
P. carabi from either Gamlingay or Waresley Woods were allowed to choose between one of the following field-collected burying beetle species: N. vespilloides, N. humator, N. interruptus and N. investigator.Each species was represented by one individual, drawn at random from a pool of field beetles (184 N. vespilloides, 98 N. humator, 129 N. interruptus and 100 N. investigator).Burying beetles in this pool were haphazardly chosen from four field populations (Gamlingay Woods, Waresley Woods, Madingley Woods (Latitude: 52.22658°;

(
post hoc comparison: z = −2.86,p = .022),but carried similar number of mites compared to those of N. humator (post hoc comparison: z = −0.73,p = .886)and N. investigator (post hoc comparison: z = −1.53,p = .418).In Waresley Wood, however, N. vespilloides F I G U R E 1 Number of Poecilochirus carabi carried by each Nicrophorus spp. in Gamlingay and Waresley Woods.Nicrophorus vespilloides, Nicrophorus humator, Nicrophorus interruptus and Nicrophorus investigator were represented as ves, hum, int and inv, respectively.Means with standard error are shown, whereas individual data were pointed with a jitter effect to prevent overlap (Note that N. interruptus and N. investigator are routinely found in Gamlingay Woods, though not in Waresley Woods-see Sun et al., 2020 for further details).

F
Population differences of mite preferences between Gamlingay (G) and Waresley (W) Woods, tested over two generations.(a) Proportion of mites that were attracted to each Nicrophorus spp. in the first generation (hum, Nicrophorus humator; int, Nicrophorus interruptus; inv, Nicrophorus investigator; ves, Nicrophorus vespilloides) and (b) proportion of mites that were attracted to their parents' preferred Nicrophorus spp.P-ves, P-hum, P-int and P-inv represent mites that chose N. vespilloides, N. humator, N. interruptus and N. investigator, respectively.Means with standard error are shown.The dashed line at 25% represents the proportion of Poecilochirus carabi associating simply by chance with one of the Nicrophorus species.

|
Prediction 2: A mixture of P. carabi lineages negatively affects N.
In the mite treatments, 'mix' means that beetles bred alongside 10 mites as a mixture of all four 'lineages' and 'ves' means that beetles bred alongside 10 mites from pure Nicrophorus vespilloides lineage.Means with standard error are shown.Each point represents a different reproductive event.Burying beetle and mite reproductive success in relation to woodland of origin and mite/beetle treatments.Beetles/mites deriving originally from Gamlingay Woods are shown in blue, whereas those deriving originally from Waresley Woods are shown in red.The beetle/mite treatments refer to the origin of the partner species.Reproductive success of beetles was measured as (a) brood size, (b) brood mass and (c) average larval mass, whereas mite reproductive success was measured as (d) the number of deutonymphs dispersing with adult beetles.Means with standard error are shown.Each point represents a different reproductive event.Values < .05were highlighted in bold.
Note: p Values < .05werehighlighted in bold.Abbreviations: hum, Nicrophorus humator; int, Nicrophorus interruptus; inv, Nicrophorus investigator; ves, Nicrophorus vespilloides.Brood massWe found that different mite populations affected burying beetle fitness in different ways, depending on the burying beetle's woodland of origin (Table2).In Gamlingay Woods, we found that Gamlingay beetles breeding with Gamlingay mites produced lighter broods compared to when they bred with Waresley mites (post hoc comparison, local mites vs. foreign mites: t = −2.28,p= .024).Beetles from Waresley Woods produced broods that did not differ significantly in mass whether they were breeding alongside Waresley mites or Gamlingay mites (post hoc comparison, local mites vs. foreign mites: t = 0.76, p = .451).F I G U R E 4